For many years, chromatographic and filter separation systems have been used to isolate one or more components of a multi-component sample. For example, liquid chromatography devices typically include a column which is packed with a solid absorbent that selectively retards the movement of sample components through the column. Chromatography has been used widely for the separation of closely related organic compounds and is an indispensable step in most chemical analysis. More recently, affinity chromatography and filtration devices have evolved which utilize highly specific complimentary binding sites on the stationary and mobile phases. These systems have been used successfully for the separation and purification of biological macromolecules.
Affinity chromatography and filtration techniques now exist which can be used to selectively bind and separate complex biological materials such as cells. Typically, polyclonal or monoclonal antibodies which selectively bind to an antigenic determinant or epitope on the surface of the target cell are produced using immunization and purification techniques which will be known to those skilled in the art. The purified antibodies are then immobilized on a solid substrate which can be packed into a column or the like. A suspension containing the target cell population is then flowed through the column whereby the immobilized antibodies selectively recognize and bind the complimentary cell-surface antigen. Thus, the target cells are retained in the column, bound to the immobilized antibodies. If desired, the bound target cells may than be eluted using a suitable carrier.
It is also known that magnetic or paramagnetic beads can be coated with antibodies which selectively bind complex biological materials such as viruses and cells. The coated beads or microspheres are then used to separate and isolate a preselected component from a mixture. For example, in copending U.S. patent application Ser. No. 113,589 filed Oct. 27, 1987, which has been assigned to the assignee of the present invention, the use of paramagnetic microspheres which are coated with antibodies that selectively bind T-lymphocyte cells is disclosed in a novel detection method. In substance, T-lymphocytes are separated from a whole blood sample by placing the sample in a test tube or the like along with antibody-coated paramagnetic beads. A bond then forms between the antibody coating and the T-lymphocytes. Using a magnetic field generator, a magnetic field is generated which causes the paramagnetic microspheres to which the T-cells are attached to adhere to the walls of a test tube. Other blood constitutents may then be removed simply by aspirating the contents of the tube while the paramagnetic microspheres and the attached T-cells remain magnetically adhered to the tube walls. This separation technique is used to isolate a specimen in which a pathogen can then be detected. In one embodiment, the aforementioned detection method is utilized to detect the presence of the virus responsible for autoimmune deficiency syndrome (AIDS).
In recent years, the proliferation of certain diseases in man and animals such as AIDS has stimulated intense research efforts to develop an effective vaccine and treatment. While the aforementioned detection method provides a rapid and accurate method of detecting AIDS, current treatment modalities such as antiviral drugs have shown only limited success. By some estimates AIDS has reached epidemic proportions. Hence, the AIDS virus, referred to by several names, including lymphadenopathy-associated virus (LAV), human immunodeficiency virus type one (HIV-1) and human T-cell lymphotrophic virus type 3 (HTLV-III), has been studied extensively.
It is now known that HIV-1 is a retrovirus having a characteristic envelope protein, glycoprotein 120. Glycoprotein 120 (gp120) is linked by a transmembrane glycoprotein (gp41) to protein 18 which in turn encloses the viral capsid (protein 24) which houses viral RNA and reverse transcriptase. HIV-1 preferentially infects helper-inducer T-lympocytes by recognizing a receptor site provided by a surface antigen known as CD4 or T4 and then binding to these T4+lymphocytes. HIV enters the target cell, losing its envelope and releasing the contents of its capsid into the cell cytoplasm. The virus replicates within the infected lymphocyte and new viruses are formed which bud from the cell memberane. It has been demonstrated that gp120 is uniformly expressed by HIV and that substantially all infected subjects produce antibodies to gp120. It has also been shown that those portions of gp120 which recognize and bind T4 cells are highly conserved. Moreover infected T4+lymphocytes express gp120 on their surfaces, providing an important molecular marker for HIV infected T4 lymphocytes. Other cells such as monocytes have also been shown to include T4+subpopulations which bind to HIV gp120, although the precise role of HIV-infected monocytes in AIDS is still unclear.
It would be desirable to provide a method and apparatus by which a preselected virus, virally infected cells, or other pathogenic agent could be removed from a fluid such as blood to reduce or eliminate host infection. It would also be desirable to provide such a method and apparatus which would operate on a continuous flow basis. The present invention achieves these goals.